Obesity causes a group of serious diseases collectively known as metabolic syndrome. These obesity-related diseases include insulin resistance (type 2 diabetes mellitus), atherogenic dyslipidemia (leading to cardiovascular diseases), hypertension (risking stroke, heart attacks and chronic kidney failure) and fatty liver. A strong association between type 2 diabetes mellitus and obesity has helped coin the term “diabesity”. Obesity is one of the most critical and widespread health issues, affecting more than 400 million people worldwide. Rates of obesity in developing countries have tripled in the last 20 years, while American obesity rates are the highest in the world. 64 percent of United States adults are overweight or obese, and about one-third of American children and adolescents are overweight or obese. There is no shortage of “treatments” for obesity, including prescription and over-the-counter medications, weight loss programs, diets and exercise regimens. Some of these solutions work, but they almost categorically result in unmet expectations for both doctors and patients, with no long-term improvement in weight or overall health. The few prescription medications currently available work to centrally suppress appetite or to block fat absorption (as anti-nutrients). However, these therapies suffer from limited efficacy and/or various adverse effects. Most patients on these therapies rebound and continue to gain weight.
Obesity is not just the result of imbalanced energy intake over expenditure. Besides host genetics, diet and exercise, the gut microbiota has emerged as a key environmental factor in the development of obesity and metabolic syndrome (i.e. in genetically obese ob/ob mice) with changes of the gut microbiota in the relative abundance of the two dominant bacterial divisions (the Bacteroidetes and the Firmicutes) (Ley et al., 2005) and an increased capacity to harvest energy from the diet. The obesity trait is also transmissible or infectious: colonization of germ-free mice with an “obese microbiota” results in a significantly greater increase in total body fat than colonization with a “lean microbiota” (Turnbaugh et al., 2006), while germ-free mice are resistant to high-fat-induced diabesity (Backhed et al., 2004; Backhed et al., 2007; Rabot et al., 2010). More importantly, lipopolysaccharide (LPS) produced by Gram-negative bacteria of the gut microbiota plays a triggering role in diabesity via “metabolic endotoxemia”:high-fat diet increases not only the proportion of LPS-containing bacteria but also intestinal permeability for LPS (Cani et al., 2007). Accordingly, known microbiota modulators/gut barrier enhancers which confer general health benefits to the host animal, such as probiotics (Lee et al., 2006; Ma et al., 2008; Aronsson et al., 2010; Kadooka et al., 2010; Kang et al., 2010; Kondo et al., 2010; Chen et al., 2011; Delzenne et al., 2011; Mozaffarian et al., 2011; Fak and Backhed, 2012; Ji et al., 2012; Teixeira et al., 2012) and prebiotics (Keenan et al., 2006; Zhou et al., 2008; Zhou et al., 2009), have showed promise as new clinical tools in this specific therapeutic area of obesity and metabolic syndrome, though issues such as dietary changes undesirable to humans (i.e. >8% resistant starch prebiotic in diet) may limit the usefulness of these methods. However, in keeping with the long-held concept of intestinal immune tolerance and undermining a previous theory from (Vijay-Kumar et al., 2010), (Ubeda et al., 2012) have recently found that the anti-infective innate immunity controlled by the pathogen-pattern-sensing toll-like receptors (TLRs) seems unable to overcome the tolerance to target the obesogenic symbiotic commensal microbes in an “obese microbiota” as foreign infectious pathogens and induce a leanogenic microbiota to prevent obesity and metabolic syndrome, depriving the basis for speculating an anti-diabesity use of an anti-infective innate immunomodulator.
Currently, the only approved effective treatment for obesity is Gastric Bypass Surgery. In normal digestion, food passes through the stomach and enters the small intestine, where most of the nutrients and calories are absorbed; it then passes into the large intestine (colon), and the remaining waste is eventually excreted. In a prototypical Roux-en-Y Gastric Bypass, the stomach is made smaller by creating a small pouch at the top of the stomach using surgical staples or a plastic band; the smaller stomach is connected directly to the middle portion of the small intestine (jejunum), bypassing the rest of the stomach and the upper portion of the small intestine (duodenum). In addition to appetite suppression and a typical weight loss of 20 to 30 kg, which can be maintained for up to 10 years (Maggard et al., 2005), Gastric Bypass completely resolves type 2 diabetes mellitus within days after the surgery and well before significant weight loss. However, Gastric Bypass Surgery is restricted to the extremely obsessed due to high surgical cost, a significant mortality rate due to complications at about 1%, a failure rate at about 15%, irreversibility, gallstones, malabsorption-caused lean mass loss and a requirement of nutritional supplementation. The underlying anti-diabesity mechanism for Gastric Bypass is believed to be diverting undigested nutrients to the mid- and lower-GI track to stimulate secretion of appetite-suppressing anti-diabesity GI peptide hormones such as peptide YY (PYY), glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM) and cholecystokinin (CCK) by nutrient-sensing enteroendocrine cells (Geraedts et al., 2009; Geraedts et al., 2010; Laferrere et al., 2010; Peterli et al., 2012). Attempts to use these anti-diabesity hormones as injectable mono-therapeutics to treat obesity have been unsuccessful likely due to a need to simultaneously administer more than one hormone (Field et al., 2010). Interestingly, oral taste receptor cells display great functional similarities (in receptor expression and GI hormone production) to GI enteroendcrine cells (Wu et al., 2002; Dyer et al., 2005; Bogunovic et al., 2007; Palazzo et al., 2007; Wang et al., 2009), and (Acosta et al., 2011) showed that PYY delivered to the oral cavity of DIO mice induced fairly good reductions of food intake and body weight likely via interaction with the specific Y2 receptor on the fibers of afferent taste nerves in oral mucosa (also see U.S. Ser. No. 13/145,660). So far there are no known agents or methods capable of stimulating production of multiple GI hormones in the oral taste receptor cells to confer anti-diabesity effects.
The Gastric Bypass Surgery and (adverse effect-prone) anti-nutrient strategies have significant mechanistic overlap in up-regulating GI hormones, as anti-nutrients (such as dirlotapide) inhibit nutrient absorption to induce secretion of GI hormones (Wren et al., 2007).
The Gastric Bypass Surgery and the microbiota modulation/gut barrier enhancement anti-diabesity therapeutics (prebiotics and probiotics) also have significant mechanistic overlap in up-regulation of the GI hormones. Colonic fermentation of the anti-diabesity prebiotic, non-digestible resistant starch, liberates short-chain fatty acids to cause day-long sustained secretion of PYY and GLP-1 (Keenan et al., 2006; Zhou et al., 2008; Zhou et al., 2009). TLR agonists such as the TLR2 agonist lipoprotein/lipopeptide (Sturm et al., 2005), the TLR5 agonist flagellin (Schlee et al., 2007; Troge et al., 2012) and the TLR9 agonist CpG DNA (Lammers et al., 2003; Menard et al., 2010; Zhong et al., 2012) are important contributory factors to the beneficial effects of a probiotic. TLR agonists also have non-immunomodulatory functions of directly inducing the enteroendocrine secretion of GI hormones (which have no reported activities to enhance anti-infective innate immunity against pathogen challenges). Enteroendocrine cells such as the STC-1 cells express functional TLRs including TLR2 (Bogunovic et al., 2007). Activation of TLR4, TLR5 or TLR9 (with respective agonist LPS, flagellin or CpG oligo DNA) induces the secretion of the GI hormone CCK from STC-1 enteroendocrine cells as well as in C57BL/6 mice (Palazzo et al., 2007), implying a TLR-mediated anti-diabesity enteroendocrine mechanism for probiotics. In addition, saturated fatty acids are agonists for both TLR4 and TLR2 (Lee et al., 2001; Lee et al., 2004), thus these two TLRs may play direct nutrient-sensing roles for the most diabeisty-relevant nutrients in enteroendocrine cells. However, the potential of this immunity-independent TLR-GI hormone pathway remains unexploited in the absence of a novel non-absorbable noninflammatory TLR agonist, because the above traditional inflammatory TLR agonists have to be excluded from the use as an anti-diabesity agent to prevent a potentially harmful TLR-mediated systemic inflammatory response (as mentioned above, LPS can actually induce metabolic syndrome). Therefore, there is the need in the art of novel therapeutics for obesity and metabolic syndrome.
Brevibacillus texasporus (e.g., ATCC PTA-5854) is a previously identified soil bacterium that expresses a non-ribosomal peptide synthetase (NRPS, encoded by the operon under GenBank accession number AY953371) to produce a family/mixture of related cationic NRP variants of 13 amino acid residues (“BT peptides” or “BT lipopeptides” in light of their newly resolved N-terminal structure, and the two terms are interchangeable in the present disclosure), among which BT1583 is the most abundant variant (WO/2005/074626). The cationic peptides (as a mixture or individual peptides isolated from B. texasporus) display a broad-spectrum antibacterial activity in vitro (BT Function #1). The high degree of 16S rDNA sequence identity (98.5%) between PTA-5854 and the Brevibacillus laterosporus type strain classifies Brevibacillus texasporus as a subspecies of Brevibacillus laterosporus, with Brevibacillus laterosporus subsp. texasporus defined as Brevibacillus laterosporus strains that produce the nonribosomal peptides from the BT NRPS (or BT peptides). Genomic sequencing of at least two B. laterosporus strains (LMG 15441 and GI-9) has validated this taxonomy. Both genomes (published respectively under GenBank accession number AFRV00000000 and EMBL accession numbers CAGD01000001 to CAGD01000061) contain an intact BT NRPS operon with 99% DNA sequence identity to AY953371, even though these B. laterosporus strains are not known to be producers of BT peptides. “Brevibacillus texasporus”, “Brevibacillus laterosporus subsp. texasporus” and “B. texasporus” are thus synonymous.
The exact identity of the N-terminal residue (including its modification) of the BT peptides was unknown, and WO/2005/074626 provided a tentative N-terminal assignment of a doubly methylated Bmt, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine and an overall BT1583 structure of Me2-Bmt-Leu-DOrn-Ile-Val-Val-DLys-Val-DLeu-Lys-DTyr-Leu-Vol, in which Orn stands for ornithine and Vol stands for valinol. Though BT peptides were discovered as antibiotics based on their antibacterial activities in vitro, (WO/2005/074626 and (Wu et al., 2005)), orally delivered BT peptides (as a mixture isolated from B. texasporus) lack antibacterial activity in vivo. For example, vancomycin-resistant enterococci (VRE) are highly sensitive to the BT peptides in vitro, but orally delivered BT peptides at concentrations well above the minimal inhibition concentration fail to decolonize VRE in the mouse GI track (Kogut et al., 2007). Importantly, orally delivered BT peptides are neither digested nor absorbed in the GI track, likely due to the presence of D-form amino acid residues and their relative highly molecular weights at about 1,600 daltons respectively. Nevertheless, orally delivered BT peptides (also as a mixture isolated from B. texasporus) can cause a number of beneficial systemic effects to animals. Orally delivered BT peptides are effective in preventing respiratory colibacillosis (air sac E. coli infection) and promoting growth and increasing feed conversion in young chickens (Jiang et al., 2005). Perhaps more importantly, the in vivo anti-infective effects of BT peptides appear to be independent of the in vitro antibiotic activities, as orally delivered BT peptides are effective in preventing infections in chickens by E. coli and Salmonella at concentrations below the in vitro minimal inhibition concentrations (Jiang et al., 2005; Kogut et al., 2007; Kogut et al., 2009). It is also discovered that circulating heterophils and monocytes are primed (rather than activated) in BT-fed chickens, pointing to innate immunity modulation as a likely mechanism of action for these in vivo anti-infective effects. Considering the fact that orally delivered BT peptides travel through the GI track without being digested or absorbed, BT peptides may stimulate intestinal epithelial cells to secret factors into the blood which in turn prime leukocytes to enhance anti-infective immunity (BT Function #2).
Bogorols are a family of 5 lipopeptide antibiotics isolated from a marine bacterium Brevibacillus laterosporus PNG276 found in a Papua New Guinea tubeworm (U.S. Pat. No. 6,784,283), which also produces a number of other antibiotics, including the lipopeptide antibiotic Tauramamide (Gerard et al., 1999; Desjardine et al., 2007). The most abundant variant bogorol A has a reported molecular weight of 1,584 and a reported structure (SEQ ID No: 1) of Hmp-EDhb-Leu-DOrn-Ile-Val-Val-DLys-Val-DLeu-Lys-DTyr-Leu-Vol, in which Hmp stands for 2-hydroxy-3-methylpentanoic acid and Dhb stands for 2,3-dehydro-2-aminobutyric acid (Barsby et al., 2001; Barsby et al., 2006). As a prelude to the present invention, the inventor discovered that BT peptides are lipopeptides: BT1583 has the same structure as bogorol A, the BT and bogorol families share four common NRP members, and thus Brevibacillus laterosporus PNG276 is also a B. texasporus strain (Examples 1 and 2).